Metastatic disease remains a major cause of mortality in breast cancer and previous studies have shown that arylamine N-acetyltransferase 1 (NAT1) is associated with increased cancer spread and poorer outcomes in breast cancer patients. However, the mechanism remains largely unknown. NAT1 alters cancer cell survival and invasive capacity in vitro. Since breast cancer cell invasion has been associated with increased matrix metalloproteinase 9 (MMP9) expression, we investigated the effects of NAT1 on MMP9 expression and activity in a range of cancer cells.
NAT1 gene deleted cell-lines (Breast cancer: MDA-MB-231, T47D; Colon cancer: HT29; Cervical cancer: HeLa; Prostate cancer: 22Rv1) were generated using CRISPR/Cas9 technology. In vitro invasion was determined by fluorescent gelatin degradation assay on coverslips, visualised by confocal microscopy and quantified using ImageJ. Gene expression was quantified by real-time RT-PCR and protein expression in cells and growth medium was determined by Western blot analyses. siRNA was used to silence Hif-1α expression. ChIP-qPCR was performed to measure acetylation level of histones at specific motifs in the MMP9 promoter.
NAT1 deletion (KO) resulted in the up-regulation of MMP9 mRNA and protein level in the highly invasive MDA-MB-231 cells as well as T47D cells but not in the poorly invasive lines HT-29, HeLa or 22Rv1. Activity of the MMPs in a gelatin degradation assay showed an 8-fold increase in the MDA-MB-231 KO compared to parent cells. Hif-1α, a transcriptional factor for several MMPs, also increased significantly following NAT1 deletion. These changes were rescued by ectopic expression of NAT1 in the MDA-MB-231 KO cells. Inhibition of Hif-1α with siRNA decreased MMP9 mRNA and protein levels. ChIP-qPCR analyses on the MMP9 promoter region revealed that both acetyl-histone H3 (AcH3) and acetyl-histone H4 lysine12 (AcH4K12) levels increased on the SMYD3 binding site, but not on the proximal or distal AP-1 binding sites.
NAT1 negatively regulates MMP9 transcription via HIF1-α and by altering histone acetylation level at the SMYD3 binding site. These observations may, in part, explain the poor outcome of breast cancer patients with low or negligible NAT1 expression.
University of Queensland.
National Health and Medical Research Council of Australia.
All authors have declared no conflicts of interest.